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Summary Staphylococcus aureus is a leading cause of hospital-associated and, more recently, community-associated infections caused by highly virulent methicillin-resistant strains (CA-MRSA). S. aureus survival in the human host is largely defined by the ability to evade attacks by antimicrobial peptides (AMPs) and other mechanisms of innate host defence. Here we show that AMPs induce resistance mechanisms in CA-MRSA via the aps AMP sensor/regulator system, including (i) the d-alanylation of teichoic acids, (ii) the incorporation of lysyl-phosphatidylglycerol in the bacterial membrane and a concomitant increase in lysine biosynthesis, and (iii) putative AMP transport systems such as the vraFG transporter, for which we demonstrate a function in AMP resistance. In contrast to the aps system of S. epidermidis, induction of the aps response in S. aureus was AMP-selective due to structural differences in the AMP binding loop of the ApsS sensor protein. Finally, using a murine infection model, we demonstrate the importance of the aps regulatory system in S. aureus infection. This study shows that while significant interspecies differences exist in the AMP–aps interaction, the AMP sensor system aps is functional and efficient in promoting resistance to a variety of AMPs in a clinically relevant strain of the important human pathogen S. aureus.

Synaptosome-enriched preparations from rat and guinea pig brain tissue vigorously accumulated [3H]-adenosine 5'-monophosphate ([3H]AMP). When the accumulation of [3H]AMP was determined using incubation periods of 30 s or less, high concentrations of adenosine, dipyridamole and soluflazine did not inhibit the accumulation of label appreciably. The accumulation of [3H]AMP was saturable, temperature-dependent, osmotic-sensitive and exhibited structural specificity. Based on the kinetics of uptake by different subcellular fractions, and the inhibitory effects of other nucleotides, the uptake of AMP appeared to be mediated by three saturable systems with Kt values of approximately 0.2, 6, and 100 microM. The transport system with the highest affinity for AMP was selectively inhibited by guanosine 5'-monophosphate, and its Vmax was several fold higher in a myelin-enriched fraction than in synaptosome-enriched fractions. The transport system with the Kt approximately 6 microM was selectively inhibited by alpha, beta-methylene adenosine diphosphate, and its Vmax was several times higher in a fraction enriched in high-density synaptosomes than in fractions enriched in low-density synaptosomes or myelin. Both of these transport systems were potently inhibited by ATP and ADP. Nucleotides that were either weak or inactive as inhibitors of AMP transport included 3'-AMP, cyclic AMP, guanosine 5'-diphosphate, and the 5'-mononucleotides of cytosine, inosine, and uridine. GTP consistently enhanced uptake at concentrations greater than or equal to 1 microM. The transport of AMP was not Na(+)-dependent and was not inhibited by membrane depolarization. This transport system may mediate the release of AMP for subsequent conversion to adenosine extracellularly.

The controversial subject of the subcellular location of myocardial adenosine production was studied employing density gradient fractionation of heart muscle combined with a novel method for analyzing distribution profiles based on multiple regression (correlation) analysis. Bungarotoxin binding, N -acetyl-β- d -glucosamidase, cytochrome c oxidase, NADPH-dependent cytochrome c reductase and lactate dehydrogenase were used as markers for the plasma membrane, lysosomes, mitochondria, sarcoplasmic reticulum and cytosol, respectively. The normalized distribution frequencies (fraction of total) of 5′-nucleotidase in mitochondria, lysosomes, plasma membranes, sarcoplasmic reticulum and cytosol in the 50 × g supernatant of total homogenate of heart muscle were found to be 0, 0.25, 0.44, 0.08 and 0.23, respectively. To increase the resolution power of this approach with respect to mitochondria, a crude mitochondrial fraction was also studied, in which the normalized distribution of 5′-nucleotidase in the homogenate was 0, 0.16 and 0.84 in mitochondria, plasma membranes and lysosomes, respectively. This mainly lysosomal 5′-nucleotidase activity was 61% inhibited by the α,β-methylene analog of ADP, indicating that although the latter has been considered specific to the plasma membrane enzyme, it also inhibits the lysosomal enzyme. The intercellular distribution of 5′-nucleotidase was not studied, but the lack of this enzyme in the mitochondria indicate that the adenosine production observed during mitochondrial AMP production, e.g. during acetate oxidation in intact heart muscle, must involve AMP transport out from the mitochondria.